Methods, reagents and kits for detecting minimal residual disease

Abstract

The invention relates to the field of minimal residual disease (MRD) diagnostics, which is progressively more applied for the evaluation of treatment effectiveness in patients with a hematological malignancy, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell chronic lymphocytic leukemia (B-CLL), and multiple myeloma (MM). Provided are unique reagent compositions with carefully selected and thoroughly tested combinations of antibodies, for ≥8-color flow cytometric stainings as well as for 10-color and 12-color flow cyometric stainings, which can reach sensitivities of at least 10−4, even down to 10−5. Also provided are diagnostic kits and methods for detecting MRD.

The invention claimed is: 
     
       1. A diagnostic kit, comprising two 8-color reagent compositions for flow cytometric detection of multiple myeloma (MM) or plasma cell disorders (PCD) in a human subject, the 8-color reagent compositions comprising distinct panels fluorochrome-conjugated antibodies directed against the following combinations of markers:
 (i) CD138, CD27, CD38, CD56, CD45, CD19, CD117 and CD81; and 
 (ii) CD138, CD27, CD38, CD56, CD45, CD19, CyIgκ and CyIgλ. 
 
     
     
       2. The kit according to  claim 1 , wherein for each of the panels the following combination of fluorochromes is used: (1) pacific blue (PacB), brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO), Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3) fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE), (5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine (APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750 or Alexa700. 
     
     
       3. The kit according to  claim 1 , wherein the antibody against CD138 is conjugated to PacB, BV421 or HV450; the antibody against CD27 is conjugated to HV500 or PacO; the antibody against CD38 to FITC; the antibody against CD56 to PE; the antibody against CD45 against PerCPCy5.5; the antibody against CD19 to PECy7; the antibody against CD117 and the antibody against CyIgκ to APC; and the antibody against CD81 and the antibody against CyIgλ to APCH7, APCA750 or APCC750. 
     
     
       4. A 10-color reagent composition for flow cytometric detection of multiple myeloma (MM) or plasma cell disorders (PCD) in a human subject comprising distinct fluorochrome-conjugated antibodies directed against the markers CD138, CD27, CD38, CD56, CD45, CD19, CD117, CD81, CyIgκ and CyIgλ. 
     
     
       5. A 12-color reagent composition for flow cytometric detection of multiple myeloma (MM) or plasma cell disorders (PCD) in a human subject comprising distinct fluorochrome-conjugated antibodies directed against the markers CD138, CD27, CD38, CD56, CD45, CD19, CD117, CD81, CyIgκ, CyIgλ, CD229 and CD28. 
     
     
       6. A multi-color flow cytometric method for detecting multiple myeloma (MM) or plasma cell disorders (PCD) in a biological sample comprising cells, preferably lymphocytes, comprising the steps of:
 (i) staining the sample with the two 8-color reagent compositions of any one of  claims 1 - 3 , or the 10-color reagent composition of according to  claim 4 , or the 12-color reagent composition of  claim 5 ; 
 (ii) subjecting the sample to flow cytometry; 
 (iii) gating on cells for expression of the selected markers detected by the antibodies present in the reagent compositions; and 
 (iv) distinguishing between normal and malignant cells, based on the expression profile of the multiple markers, wherein CD38, CD27, CD45, CD19 and CD81 are underexpressed in malignant cells compared to normal cells, wherein CD56, CD28 and CD117 are overexpressed in malignant cells compared to normal cells, and wherein expression of CyIgκ and CyIgλ is restricted to either one or the other Ig light chains in malignant cells. 
 
     
     
       7. The method according to  claim 6 , wherein step (iv) involves multivariate analysis, preferably principal component analysis (PCA). 
     
     
       8. The method according to  claim 6 , wherein the biological sample is a peripheral blood sample or a bone marrow sample. 
     
     
       9. The method according to  claim 7 , wherein the biological sample is a peripheral blood sample or a bone marrow sample.